PROJECT SUMMARY Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are linked by clinical, neuropathological and genetic overlap1. It was recently discovered that a G4C2 repeat expansion in C9ORF72 is the most common genetic cause of FTD and ALS (?c9FTD/ALS?)2, 3. A growing body of evidence suggesting that repeat-containing RNA [r(G4C2)exp or r(G2C4)exp], which is bidirectionally transcribed from the C9ORF72 expansion, is a key player in c9FTD/ALS pathogenesis14. Currently, there are no effective treatments for FTD or ALS, in part because of an incomplete understanding of the causative pathological mechanisms. Herein we propose to develop small molecule chemical probes of r(G4C2)exp and r(G2C4)exp (dys)function to study repeat biology, thereby identifying the most therapeutically relevant targets and pathways to treat c9FTD/ALS. Indeed, this grant application brings forward novel and innovative chemical approaches to develop highly selective chemical probes that target RNA, to validate the RNA targets of small molecules (with Core B), to study cellular selectivity (with Core B), and to image RNA in cellular systems. Not only will our findings benefit c9FTD/ALS patients, they will have broad implications for many microsatellite expansion diseases. Our proposed studies are enabled by our recently reported studies in which we discovered three compounds that bind r(G4C2)exp in cells and significantly inhibit two modes of RNA toxicity (generation of toxic proteins via repeat-associated non-ATG (RAN) translation and formation of foci) in c9FTD/ALS in cells over-expressing r(G4C2)66 and induced neurons (iNeurons) directly converted from fibroblasts of C9ORF72 repeat expansion carriers12. In particular, we will: (i) lead optimize our previously identified compounds in collaboration with Core B; (ii) define features in small molecules that provide an ideal chemical probe of repeat (dys)function; and (iii) use our chemical probes to uncover the underlying pathophysiology of the c9FTD/ALS repeat expansion, including the mechanism of RAN translation, identification of all proteins sequestered in foci, and studying defects in nucleocytoplasmic transport of C9ORF72 by imaging the RNA in its natural context with small molecule probes (Core B). Our studies are highly synergistic with Project 2 (development of biomarkers against c9RAN proteins and studying pathophysiology in a recently developed mouse model of c9ALS/FTD13) and Project 3 (abnormalities in nuclear pores and transport caused by the c9FTD/ALS repeat expansion). Indeed, our optimal chemical probes will be provided to Profs. Petrucelli, Gendron, & Rothstein to augment their studies.